Detection of low concentration samples is of interest in many fields. Currently, much effort has been devoted to developing techniques to be able to separate a sample into its components, e.g., capillary zone electrophoresis (CZE), liquid chromatography (LC), and super critical fluid chromatography (SFC). These techniques are able to resolve a minute sample into individual components. There is a need for a very sensitive detector to measure the concentration of these microscopic quantities in samples. In the above techniques, CZE, LC, SFC, and other techniques such as flow cytometry, it is desirable to have detectors which are extremely sensitive but which are also universal in the sense that they can respond to a wide variety of materials. The detector must respond to changes in some characteristic, e.g., optical property, conductivity, etc., of the sample for concentration measurement.
Numerous techniques and apparatus, such as UV and visible photometers, mass spectrometry, refractive index, fluorescence and others, have been used to detect concentrations of materials in fluid samples. However, some of these techniques which are extremely sensitive are not universal, and some which are universal are not very sensitive. Also, some detectors require a large amount of sample in order to be able to respond. Some of the presently available detectors may not function well where a large background is present or the time response of the detector may not be compatible with the sample. Further, some of the techniques are cost prohibitive and complex.
The mass spectrometer is one of the few detectors which has both very high sensitivity and a kind of universality. However, the cost is often prohibitive for many users and, therefore, an alternative must be sought. The use of mass spectrometry also requires the transfer of material to a high vacuum chamber. The requirement for a high vacuum chamber further increases the cost and complications for the user.
As mentioned, it is desirable to have a highly sensitive detector for use with various separation techniques. There are several kinds of separation techniques, including gas and liquid chromatography. All of the chromatography techniques require the use of a detection system to aid the scientist. As an example, liquid chromatography allows separation of an injected material or solute according to size or chemical properties. The solute is injected into a suitable solvent or liquid. The individual chemical compounds originally present in a mixture are resolved from each other by the selective process of distribution between two heterogeneous phases. The separation occurs between two phases known as the stationary phase and the mobile phase. The stationary phase is a dispersed medium through which the mobile phase is allowed to flow. The greater the attraction of a particular chemical compound for the stationary phase, the longer it will be retained in the system. This retention provides separation. All types of chromatography may be defined as separation processes where components are selectively retained by a stationary phase. The components are retained to different degrees and, therefore, samples may be separated based on different physical characteristics. Another separation technique which is currently used is capillary zone electrophoresis. Electrophoresis involves migration of macromolecules or colloids in a liquid due to the effect of potential difference across immersed electrodes. The molecules or colloids migrate at different rates to provide discrete zones. The molecules or colloids travel from the inlet of a capillary to the output, where they are detected as they approach the exit.
Yet another example of a technique which is benefitted by a sensitive detector is flow cytometry. A flow cytometer is an apparatus which sorts cells or particles. The cells or particles are passed in a single line through a laser beam which causes them to fluoresce. Sensors then measure physical or chemical characteristics of the cells or particles. More particularly, a user may wish to utilize a flow cytometer to count cells in liquid suspension or cells in aerosols.
A detector having the ultimate sensitivity would respond to a single molecule. A universal detector which responds to single molecules still requires some means to discriminate between molecules of the solute and molecules of the solvent. The present invention addresses the problems associated with measuring concentration of macromolecules and colloids in liquid samples. The apparatus of the present invention operates in an accurate, controlled, more sensitive and universal manner. The apparatus is a real time system which possesses the sensitivity and ability to detect single molecules.